The objective of this study is to investigate the structure of the procollagenase/collagenase molecule and the changes associated with the activation process. Procollagenase produced by human skin fibroblasts in vitro was purified to apparent homogenity by successive affinity chromatography on Zn++ chelate-sepharose and heparin-separose followed by Ultrogel AcA 44 molecular sieve chromatography. The resultant preparation consisted of a major lead (54K) and a minor trailing (57K) polypeptide chain. Conversion to active form either by trypsin or organomercurials resulted in generation of two new (44K/47K) components. The relationship between the various molecular forms was studied using polyclonal antibodies (PAs) against procollagenase and by a complement of monoclonal antibodies (MAs) directed against the various structural domains of the procollagenase molecule. Immunoperoxidase staining of Western blots of the various molecular forms of collagenase/procollagenase resolved by SDS-PAGE revealed two additional minor components both in the procollagenase and the collagenase peptide groups. Each of the components generated during the activation reaction formed SDS-stable complexes with Alpha2-macroglobulin and therefore had acquired catalytic activity. A single MA (Clone X2a) reacted only with the procollagenase species and not with the active forms which leads us to believe that the procollagenase molecule possesses a domain that is lost during the activation reaction, possibly an activation peptide.